We have focused on those aspects of eukaryotic DNA replication that appear to be unique to the metazoa: how do the metazoa regulate the number and locations of initiation sites during cell proliferation and development? We have previously mapped origins of bi-directional replication (OBRs) to specific chromosomal sites of 0.4 to 2 kb in the hamster genome. We have now been able to demonstrate that pre-replication complexes are assembled at or close to these sites when hamster cells transit from metaphase to G1 phase in their cell division cycle. These pre-RCs can be activated in vitro using a Xenopus egg extract. In eukaryotes, site specificity is determined by a six member ?origin recognition complex? that binds to specific sites along the genome. Fission yeast (S. pombe), like mammals, contain large, AT-rich replication origins that lack a recognizable consensus sequence, but that nevertheless contain sequences required for replicatioin. We have discovered that the SpOrc4p alone bound tightly and specifically to several sites within S. pombe replication origins that are genetically required for origin activity. These sites consisted of clusters of A or T residues on one strand, but were devoid of either alternating A and T residues or GC-rich sequences. Addition of a complex consisting of Orc1, 2, 3, 5 and 6 proteins (ORC-5) did not alter either Orc4p binding to origin DNA, or Orc4p protection of specific sequences. ORC-5 alone bound weakly and non-specifically to DNA; strong binding required the presence of Orc4p. Under these conditions, all six subunits remained bound to chromatin isolated from each phase of the cell division cycle. These results reveal that the S. pombe ORC binds to multiple, specific sites within replication origins, and that site selection, at least in vitro, is determined solely by the Orc4p subunit. We have further shown that Orc4p binds specifically to only one of the four required sequences in ARS3001 where it initiates assembly of a pre-RC and initiates bi-directional DNA replication. Thus, S. pombe replication origins may provide an appropriate paradigm for replication origins in higher eukaryotes. In mammals, we have identified the existence of a novel regulatory pathway in the initiation of DNA replication. Orc1 and Orc2 are both tightly bound to chromatin during late G1?phase, and late G1?phase nuclei contain active ORC/chromatin sites by virtue of the fact that they can initiate DNA replication at specific genomic sites when incubated in an Orc?depleted Xenopus egg extract. In contrast to yeast where all six ORC subunits are stably bound to chromatin throughout the cell cycle, the affinity of mammalian Orc1 for chromatin is selectively reduced during S-phase, such that lysis of cells in 0.1% Triton X-100, 0.15 M NaCl, and 1 mM Mg++ATP releases Orc1, but not Orc2, into the chromatin unbound fraction. Moreover, the Orc1 that is released during S-phase is rapidly ubiquitinated with only one or two ubiquitin adducts. In contrast, Orc2 is not a substrate for ubiquitination. During the S to M transition, Orc1 is deubiquitinated. During the M to G1 transition, Orc1 rebinds tightly to hamster ORC/chromatin sites to allow assembly of pre-replication complexes. This sites are located at specific genomic loci referred to as ?origins of bi-directional replication?. The role of Ubiquitination is to sequester Orc1 during S-phase, and thus prevent reinitiation at replication origins during a single cell division cycle. However, if Ub-Orc1 is released into the cytosol, then it is polyubiquitinated and degraded by the 26S proteasome pathway. This provides a mechanism for reprogramming replication origins during animal development or as a result of DNA damage. Thus, in contrast to yeast, mammalian ORC activity appears to be regulated during each cell cycle through selective dissociation and reassociation of Orc1 from chromatin bound ORC. In searching for the trigger that releases Orc1 from mammalian chromatin, we incubated mammalian mitotic chromatin (which lacks functional ORCs) in a Xenopus egg extract to initiate DNA replication. We discovered that the entire Xenopus ORC rapidly binds to the somatic cell chromatin, initiates DNA replication, and is released as soon as Mcm proteins are bound to form a pre-RC.